Nucleic acid hybridization assays are commonly used in genetic research, biomedical research and clinical diagnostics. In a basic nucleic acid hybridization assay, the nucleic acid of interest is hybridized, in single-stranded form, to a labeled single-stranded nucleic acid probe and resulting labeled duplexes are detected. Variations of this basic scheme have been developed to enhance accuracy, facilitate the separation of the duplexes to be detected from extraneous materials, and/or amplify the signal that is detected.
Commonly assigned U.S. patent application Ser. No. 07/559,961, filed Jul. 27, 1990 now U.S. Pat. No. 5,430,136 and incorporated by reference herein, describes a technique whereby selectably cleavable sites are introduced into oligonucleotide chains, enabling release of a detectable label after hybridization is complete. As explained in that application, selectably cleavable sites are useful in a number of different types of hybridization assay formats. For example, in one type of assay in which hybridization gives rise to a solid-supported duplex of a labeled probe and sample DNA, a selectably cleavable site contained within the hybrid structure will enable ready separation of the label from the solid support. Commonly assigned U.S. Pat. Nos. 4,775,619 and 5,118,605 are respectively directed to the use of restriction endonuclease cleavable sites in such assays and the use of chemically cleavable sites (e.g., disulfide linkages, 1,2-diols, and the like). These cleavable sites can be introduced during oligonucleotide synthesis, and are cleavable with restriction endonucleases in the case of restriction sites and with particular chemical reagents, e.g., with thiols, periodate, or the like, in the case of chemically cleavable sites.
The present invention is also directed in part to the incorporation of selectably cleavable sites into polynucleotides. The cleavable sites herein are contained within a linker arm present at the 1 position of a deoxyribose molecule. In addition to providing such aleavable sites, the invention also relates to the creation of "abasic sites" within polynucleotides, i.e., monomeric units which contain the deoxyribose ring but do not have a purine or pyrimidine base present at the 1 position. Such abasic sites are useful in a wide variety of contexts, as will be explained in detail hereinbelow. For example, an abasic site may be used to create branched DNA, i.e., a multimeric polynucleotide structure in which three polynucleotide chains emanate from a single deoxyribose unit. These branch points are extremely useful in providing large, "multimeric" DNA structures which can then be used in amplification assays. Abasic sites may also be used in other ways, e.g., in the synthesis of DNA bound to a solid support (typically although not necessarily at the 1 position), to reverse the direction of chemical DNA synthesis, i.e., 3.fwdarw.5' to 5.fwdarw.3' or vice versa, and in triple helix formation.
Thus, in addition to utility in providing cleavable sites within oligonucleotide or polynucleotide chains, the invention enables a number of procedures deriving from the presence of linker arms at the 1 position of a monomeric deoxyribose unit rather than purine or pyrimidine bases as present in conventional nucleotide structures.
Overview of the Art
Background references which relate generally to methods for synthesizing oligonucleotides include those related to 5'-to-3' syntheses based on the use of .beta.-cyanoethyl phosphate protecting groups, e.g., de Napoli et al., Gazz Chim Ital 114:65 (1984), Rosenthal et al., Tetrahedron Letters 24:1691 (1983), Belagaje and Brush, Nucleic Acids Research 10:6295 (1977), in references which describe solution-phase 5'-to-3' syntheses include HayatSu and Khorana, J American Chemical Society 89:3880 (1957), Gait and Sheppard, Nucleic Acids Research 4:1135 (1977), Cramer and Koster, Angew. Chem. Int. Ed. Engl, Z:473 (1968), and Blackburn et al., Journal of the Chemical Society, Part C, 2438 (1967).
In addition to the above-cited art, Matteucci and Caruthers, J. American Chemical Society 103:3185-3191 (1981), describe the use of phosphochloridites in the preparation of oligonucleotides. Beaucage and Caruthers, Tetrahedron Letters 22:1859-1862 (1981), and U.S. Pat. No. 4,415,732 describe the use of phosphoramidites in the preparation of oligonucleotides. Smith, ABL 15-24 (December 1983), describes automated solid-phase oligodeoxyribionucleotide synthesis. See also the references cited therein, and Warner et al., DNA 3:401-411 (1984), whose disclosure is incorporated herein by reference.
U.S. Pat. Nos. 4,483,964 and 4,517,338 to Urdea et al. describes a method for synthesizing polynucleotides by selectively introducing reagents to a solid phase substrate in a tubular reaction zone. U.S. Pat. No. 4,910,300 to Horn et al. also describes a method for synthesizing oligonucleotides by sequentially adding nucleotidic monomers to a growing chain, but involves the incorporation of labelled, N-4 modified cytosine residues at predetermined, spaced apart positions. U.S. Pat. No. 5,256,549 to Horn et al. is also of interest in that a method for preparing oligonucleotides is provided which involves a combination technique, i.e., in which the desired oligonucleotide is essentially synthesized and "purified" simultaneously, such that the final product is produced in substantially pure form.
Horn and Urdea, DNA 5(5):421-425 (1986), describe phosphorylation of solid-supported DNA fragments using bis(cyanoethoxy)-N,N-diisopropyl-aminophosphine. See also, Horn and Urdea, Tetrahedron Letters 27:4705-4708 (1986).
References which relate to hybridization techniques in general include the following: Meinkoth and Wahl, Anal. Biochemistry 138:267-284 (1984), provide an excellent review of hybridization techniques. Leary et al., Proc. Natl. Acad. Sci. (USA) 80:4045-4049 (1983) describe the use of biotinylated DNA in conjunction with an avidin-enzyme conjugate for detection of specific oligonucleotide sequences. Ranki et al., Gene 21:77-85, describe what they refer to as a "sandwich" hybridization for detection of oligonucleotide sequences. Pfeuffer and Helmrich, J. Biol. Chem. 250:867-876 (1975), describe the coupling of guanosine-5'-O-(3-thiotriphosphate) to Sepharose 4B. Bauman et al., J. Histochem. and Cytochem. 29:227-237, describe the 3'-labeling of RNA with fluorescers. PCT Application WO 83/02277 describes the addition to DNA fragments of modified ribonucleotides for labeling and methods for analyzing such DNA fragments. Renz and Kurz, Nucl. Acids. Res. 12:3435-3444, describe the covalent linking of enzymes to oligonucleotides. Wallace, DNA Recombinant Technology (Woo, S., ed.) CRC Press, Boca Raton, Fla., provides a general background of the use of probes in diagnosis. Chou and Merigan, N. Eng. J. of Med. 308:921-925, describe the use of a radioisotope-labeled probe for the detection of CMV. Inman, Methods in Enzymol. 34B, .24:77-102 (1974), describes procedures for linking to polyacrylamides, while Parikh et al., Methods in Enzymol. 34B, 24:77-102 (1974) describe coupling reactions with agarose. Alwine et al., Proc. Natl. Acad. Sci. (USA) 74:5350-5354 (1977), describe a method of transferring oligonucleotides from gels to a solid support for hybridization. Chu et al., Proc. Natl. Acad. Sci. (USA) 11:6513-6529, describe a technique for derivatizing terminal nucleotides. Ho et al., Biochemistry 20:64-67 (1981), describe derivatizing terminal nucleotides through phosphate to form esters. Ashley and MacDonald, Anal. Biochem 140:95-103 (1984), report a method for preparing probes from a surface-bound template.
Horne and Dervan, J. Am. Chem. Soc. 112:2435-2437 (1990), and Froehler et al., Biochemistry 31:1603-1609 (1992), relate to oligonucleotide-directed triple helix formation.